SIST EN 15708:2010

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Wasserbeschaffenheit - Anleitung zur Beobachtung, Probenahme und Laboranalyse von Phytobenthos in flachen FließgewässernQualité de l'eau - Guide pour l'étude, l'échantillonnage et l'analyse en laboratoire du phytobenthos dans les cours d'eau peu profondsWater quality - Guidance standard for the surveying, sampling and laboratory analysis of phytobenthos in shallow running water13.060.70Preiskava bioloških lastnosti vodeExamination of biological properties of water13.060.10Voda iz naravnih virovWater of natural resourcesICS:Ta slovenski standard je istoveten z:EN 15708:2009SIST EN 15708:2010en,fr,de01-februar-2010SIST EN 15708:2010SLOVENSKI
STANDARD



SIST EN 15708:2010



EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 15708
November 2009 ICS 13.060.70 English Version
Water quality - Guidance standard for the surveying, sampling and laboratory analysis of phytobenthos in shallow running water Qualité de l'eau - Guide pour l'étude, l'échantillonnage et l'analyse en laboratoire du phytobenthos dans les cours d'eau peu profonds
Wasserbeschaffenheit - Anleitung zur Beobachtung, Probenahme und Laboranalyse von Phytobenthos in flachen Fließgewässern This European Standard was approved by CEN on 10 October 2009.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the CEN Management Centre or to any CEN member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by translation under the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre:
Avenue Marnix 17,
B-1000 Brussels © 2009 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 15708:2009: ESIST EN 15708:2010



EN 15708:2009 (E) 2 Contents Page Foreword .3Introduction .41Scope .52Normative references .53Terms and definitions .54Principle .85Reagents .86Equipment .87Survey and sampling strategy . 108Survey and sampling procedures . 129Identification and basic quantification of organisms . 1710Data processing and interpretation . 1811Quality assurance . 19Annex A (informative)
Worked example of computation of final abundance estimates for a MHS from an upland stream in NE England . 20Bibliography . 21 SIST EN 15708:2010



EN 15708:2009 (E) 3 Foreword This document (EN 15708:2009) has been prepared by Technical Committee CEN/TC 230 “Water analysis”, the secretariat of which is held by DIN. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by May 2010, and conflicting national standards shall be withdrawn at the latest by May 2010. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. SIST EN 15708:2010



EN 15708:2009 (E) 4 Introduction WARNING — Working in or around water is inherently dangerous. Persons using this European Standard should be familiar with normal laboratory practice. Long periods of analysis at the microscope can cause physical fatigue and affect eyesight. Attention should be given to the ergonomics of the microscope and advice from a health and safety practitioner should be sought to ensure that risks are minimized. The use of chemical products mentioned in this standard can be hazardous and users should follow guidelines provided by the manufacturers and take necessary specialist advice. This standard does not purport to address the safety problems associated with its use. It is the responsibility of the user to establish appropriate health and safety practices and to ensure compliance with any national regulatory conditions. The phytobenthos is an important component of aquatic ecosystems and an understanding of the composition of the phytobenthos present in a waterbody can provide useful information on the status of that waterbody, and on appropriate management strategies. The Water Framework Directive (2000/60/EC) [3] requires monitoring of the phytobenthos as a quality element used for ecological status assessment, and phytobenthos assessments have also been used in monitoring programmes associated with other European Directives (e.g. Urban Wastewater Treatment Directive, Habitats Directive) and with national legislation (e.g. ÖNORM M6231). This guidance standard specifically relates to the sampling of phytobenthos (other than aquatic macrophytes) in running water. An etymologically-correct application of the term “phytobenthos” would cover all phototrophic organisms; however, this encompasses a vast range of organisms, from microscopic unicells to macrophytes > 2 m in length. As separate survey methods for aquatic macrophytes are available (EN 14184), this document focuses on phototrophic algae and oxygenic cyanobacteria that live on substrata. Bryophytes are common in shallow running waters and competitive interactions between these and larger algae are common. Similarly, aquatic macrophyte species may, themselves, act as substrata or competitors for algae and cyanobacteria. For these reasons, the standard provides options for including these taxa in survey and sampling procedures. The term “periphyton” is sometimes used instead of “phytobenthos”; however, some definitions of “periphyton” include heterotrophic organisms that live attached to substrata (protozoa, sponges, hydroides). Methods described here deal only with photosynthetic organisms but they could, if required, be adapated to encompass heterotrophic organisms too. Methods using phytobenthos to assess water quality in running water have been developed in several European countries [6], [8], [9], [10] and in the USA [2]. Recent work is summarised in the proceedings of four symposia [1], [7], [11], [12]. Methods for the sampling and analysis of one group of phytobenthos, the diatoms, have already been the subject of harmonisation (EN 13946, EN 14407). However, these standards are concerned with only a single group of the phytobenthos and there are situations where other phototrophs are more obvious and can contribute additional ecological information. According to the precise usage to which this standard is to be put it is essential for specifiers and users to mutually agree on any necessary variations or optional procedural details prior to use. SIST EN 15708:2010



EN 15708:2009 (E) 5 1 Scope This European Standard provides guidelines for the survey/sampling, identification and basic quantification of phytobenthos (other than macrophytes) in running waters. It is applicable to rivers where benthic algae and bryophytes are the main phototrophs. This method encompasses all phytobenthic growth forms and enables biological responses to environmental events over one or more years to be monitored. In this respect it provides an alternative to methods based on benthic diatoms (EN 13946; EN 14407) and macrophytes (EN 14184). Data obtained for the phytobenthos growth forms are suitable for pilot surveys, water quality assessment and trend monitoring. This European Standard encompasses all aspects from the design of survey and sampling programmes to the identification and basic quantification of the phytobenthos. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. EN 13946, Water quality — Guidance standard for the routine sampling and pretreatment of benthic diatoms from rivers EN 14407, Water quality — Guidance standard for the identification, enumeration and interpretation of benthic diatom samples from running waters EN 15204, Water quality — Guidance standard on the enumeration of phytoplankton using inverted microscopy (Utermöhl technique) 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 aquatic macrophytes larger plants of fresh water which are easily seen with the naked eye, including all aquatic vascular plants, bryophytes, stoneworts (Characeae) and macro-algal growths [EN 14184:2003, 3.1]
3.2 assemblage organisms that share a sampling area NOTE This term is preferred to “community”, as the latter implies a level of ecological integration of the organisms; whereas sampling may inadvertently combine representatives from more than one true “community” that are not distinct to the naked eye. 3.3 belt transect defined band across a river or stream at right angles to the bank NOTE This may be virtual or physically delineated within which the aquatic vegetation is analysed (species composition, abundance, cover). [EN 14184:2003, 3.4] SIST EN 15708:2010



EN 15708:2009 (E) 6 3.4 benthic alga alga or oxygenic cyanobacterium living attached to a substratum (rather than suspended in the water column) 3.5 biofilm mucilagineous polysaccharide matrix on submerged stable surfaces consisting of photo(auto)trophic and heterotrophic organisms 3.6 boulder mineral substratum with a diameter > 256 mm [EN 13946:2003, 3.3] 3.7 bryophyte collective term for liverworts and mosses – plants which are often abundant on boulders and bedrock of fast flowing streams
3.8 cobble mineral substratum with a diameter > 64 mm and ≤ 256 mm [EN 13946:2003, 3.4] 3.9 degree of cover percent of substratum at the sampling site covered (by the organism) 3.10 epilithic alga alga living attached to or in close association with a stony substratum 3.11 epiphytic alga alga living attached to or in close association with macrophytes or other algae 3.12 epipelic alga alga that lives in or on fine sediments 3.13 epipsammic alga alga that lives attached to or in close association with sand 3.14 habitat specific environment in which a species lives [EN 15460:2007, 3.5] 3.15 macroscopic benthic alga multicellular alga or an aggregation (units/groupings) of unicellular algae living attached to substrata that is visible to the naked eye SIST EN 15708:2010



EN 15708:2009 (E) 7 3.16 nuisance biomass accumulations of benthic algae that are a nuisance to users of the watercourse and/or that detrimentally affect its ecology 3.17 periphyton group of organisms (principally algae, but also including fungi, bacteria and protozoa) living on or in close contact with surfaces in aquatic environments NOTE 1 Bryophytes have an intermediate position. They are often regarded as a component of the aquatic macrophytes, particularly in slow flowing rivers where aquatic macrophytes are common. NOTE 2 The term “periphyton” is often used as a synonym for benthic algae in recent literature. 3.18 phototroph organism whose main source of carbon is obtained through photosynthesis NOTE For the purpose of this document, facultative phototrophs such as many Euglenophyta are included within this definition. 3.19 phytobenthos all phototrophic algae and oxygenic cyanobacteria living on or in close contact with surfaces in aquatic environments
NOTE This term is virtually synonymous with the term "periphyton", although some people also include heterotrophic organisms in definitions of the latter. 3.20 reach length of a watercourse forming a major sub-division of a river basin and defined by physical, chemical or hydrological characteristics (or any combination of these) that distinguishes it from the watercourse upstream and downstream NOTE The boundaries between reaches mark the principal points of transition where the overall character of the watercourse changes. [EN ISO 8689-2:2000, 3.1] 3.21 reference conditions conditions reflecting a totally undisturbed state, or one with only very slight human impacts, or near-natural with only minor evidence of distortion 3.22 riffle fast-flowing shallow water with distinctly broken or disturbed surface over gravel/pebble or cobble substratum
[EN 14614:2004, 2.28] 3.23 survey unit length of river from which data are collected during field survey; this may be a fixed length (e.g. 10 m) or variable, according to the methods used, but must always be defined and recorded [Adapted from EN 14614:2004, 2.38] SIST EN 15708:2010



EN 15708:2009 (E) 8 3.24 taxon group of organisms related at a particular taxonomic level [EN 14996:2006, 3.20] NOTE Plural is "taxa". 4 Principle Phototrophs associated with submerged surfaces in running water are surveyed and/or sampled. Specimens of those taxa that cannot be identified in the field are taken back to the laboratory for identification. Three different options are provided within the standard, suitable for different circumstances. Outcomes of the survey/sampling process may include: a) a list of all macroscopic algae (and, optionally, non-vascular plants) observed in the survey unit; b) a list of all macroscopic and microscopic algae (and, optionally, non-vascular plants) observed in the survey unit; or c) a list of all microscopic and macroscopic algae found on a single substratum within the survey unit.
Semi-quantitative estimates of the abundance of each taxon are also made. These data can be used to give an integrated picture of ecological status and/or water quality.
5 Reagents Preservatives are necessary if samples are to be stored prior to analysis. If treated with care, many algal samples can be stored in a refrigerator or cool room for several days without deterioration. However, where long-term storage is necessary, then a preservative may be necessary. Recipes for preparation of these are given in EN 15204 (see also 8.4). 6 Equipment 6.1 Field equipment 6.1.1 Necessary field equipment 6.1.1.1 Appropriate water safety equipment 6.1.1.2 A means of locating sampling reaches on repeat visits, if there is no permanent landmark adjacent. Options include iron bolts, fast drying paint, waterproof tape or similar to delimit the sampling reaches. 6.1.1.3 Waders 6.1.1.4 Aqua-scope, or bucket with clear Perspex base, for scanning the river bottom in turbulent water. 6.1.1.5 Stainless steel knife or other suitable blade, forceps and stiff toothbrush 6.1.1.6 Hand lens 6.1.1.7 White plastic or enamel tray, volume 2 l to 3 l, for sorting material and sub-sampling. SIST EN 15708:2010



EN 15708:2009 (E) 9 6.1.1.8 Sample vials with tight fitting lids, recommended sizes are 5 ml and 125 ml, to encompass both singular macroscopic units and composite samples. 6.1.1.9 Waterproof labels for sample vials, or a marker pen with waterproof ink. 6.1.1.10 Waterproof fieldbook, or standardised recording sheets plus pencil or indelible pen. 6.1.1.11 Preservative, buffered formalin, Lugol’s iodine, or other. CAUTION — The use of formalin can cause health problems. 6.1.2 Optional field equipment 6.1.2.1 Global positioning system (GPS) receiver 6.1.2.2 Rake with attached net or hoe attached to a long handle, to facilitate sampling at high flow. 6.1.2.3 Bucket, to transfer large substrata to laboratory. 6.1.2.4 Camera or video-camera 6.1.2.5 Portable refrigerator or ice box 6.1.2.6 Boxes with room, to store all sample vials from one locality, to facilitate storage. 6.2 Laboratory equipment 6.2.1 Necessary laboratory equipment 6.2.1.1 Binocular microscope, equipped with a mechanical stage and at least 40 × magnification for sorting of samples. 6.2.1.2 Compound light microscope, equipped with a mechanical stage and medium (e.g. 40 ×) and high power (e.g. 100 ×) objectives. The microscope should incorporate facilities for measurements (e.g. an eyepiece graticule) with a resolution of at least 1 µm. Use of a phase contrast or differential interference (Nomarski) condenser may be useful. 6.2.1.3 Microscope slides and cover glasses 6.2.1.4 Immersion oil, dispenser, lens papers and absorbent tissues 6.2.1.5 Floras, identification guides and iconographs (illustrations), appropriate to the habitats under consideration. 6.2.1.6 Facility for recording data, as they are collected. This can be a pro forma sheet with a list of taxa and space beside each on which the abundance estimation can be made or a laboratory notebook organised in such a way that taxon identities and abundance can be clearly recorded.
6.2.2 Optional laboratory equipment 6.2.2.1 Apparatus for photo-microscopy or digital image capture 6.2.2.2 Tissue homogenizer or blender 6.2.2.3 Magnetic stirrer and stir bar, forceps 6.2.2.4 Tally counter, for species proportional count. 6.2.2.5 Apparatus and equipment for preparing diatom samples (see EN 13946) SIST EN 15708:2010



EN 15708:2009 (E) 10 7 Survey and sampling strategy 7.1 Approaches Any reach within a river or stream is likely to contain a number of different substrata on which phytobenthos can grow, and some of these substrata may, in turn, be colonised by more than one type of phytobenthic growth form. In many cases, these growth forms will be visible to the naked eye, even if the constituent organisms are microscopic. In some cases, the growth form will contain one, or a few, dominant organism(s) along with epiphytes and loosely-associated taxa. The dominant organism(s) may be identified in the field, although it may be necessary to confirm the identity in the laboratory. In other cases (e.g. epilithic biofilms), the dominant organism may be too small to be identified in the field. The result is that any survey unit will contain a wide variety of growth forms that may need to be recorded and/or sampled. Analysis of the phytobenthos at a site/survey unit consists of three stages, which can be combined in various ways to give a number of survey/sampling strategies, each applicable to different purposes. These stages are:  Survey: a detailed inspection of a defined length of the river or stream, recording the nature of the stream environment, the substrata available for phytobenthos and the nature and abundance of any phytobenthic growth forms present.  Sampling: removal of small quantities of some or all the phytobenthic growth forms for subsequent examination in the laboratory.  Laboratory analysis: identification and abundance assessment of the organisms present in the growth forms. In a few cases (e.g. Hildenbrandia rivularis), species-level determinations can be made in the field but in most cases, the identities of macroscopic algae and bryophytes should be checked in the laboratory unless the surveyors have proven competence in field identification of these organisms. These three stages are combined, in different ways, to give the following sampling strategies:  Macroscopic Phytobenthos Survey (MPS): detailed survey of all, or a selection, of the phytobenthic growth forms that are visible with the naked eye, with sampling and laboratory analysis confined to checking the identities of macroscopic algae (and optionally bryophytes). MPS provides semi-quantitative (or, with slight modification, quantitative) estimates of the abundance of those taxa that are visible to the naked eye. It is recommended for trend monitoring and, particularly, for detecting changes in abundance of those algae such as Cladophora and Hydrodictyon which are capable of proliferating to "nuisance" quantities.  Multi Habitat Sampling (MHS): survey and sampling of all available habitats/substrata in order to compile a list, with semi-quantitative estimates of abundance, of all phytobenthic taxa present at a survey unit. MHS best characterises the phytobenthos in the reach, but results may not be sensitive to subtle water quality differences because of habitat differences between reaches.
 Single Habitat Sampling (SHS): a single type of habitat/substratum is sampled at each survey unit and examined in the laboratory. The output is, as for MHS, a list, with abundance estimates, of all taxa present. SHS should reflect water quality differences between streams more precisely than MHS, provided that the same type of habitat/substratum is sampled at all sites. Impacts on other habitats/substrata in the reach may however be missed. It is identical, in principle, to methods described in EN 13946, except that groups other than the diatoms are included in the subsequent analysis. Figure 1 is a diagrammatic representation of how these strategies can be applied to a survey unit.
SIST EN 15708:2010



EN 15708:2009 (E) 11
Habitat 1
Habitat 2
Habitat 3
…. Habitat n
Field
Assess
Assess
Assess
… Assess
cover
cover
cover
cover
MPS
Laboratory Assess
Assess
Assess
… Assess
composition
composition
composition
composition
Data
Data
Data
Data
MHS
SHS Figure 1 — Relationships between field and laboratory stages for the three types of analysis described in this European Standard 7.2 Number and location of sampling sites The number and location of sampling or survey sites should be determined according to the aim of the study. In general, sites selected should be representative of the reach of river under consideration, avoiding heavily-shaded sites unless these are characteristic of the reach. Where possible, sites selected should have similar conditions of light, current velocity, substratum, etc. to facilitate comparability. Reaches should be identified within a watercourse with respect to points where marked changes in quality are likely to occur or where there are important river uses, for example major discharges or abstractions. Sufficient sample sites should be included within each reach to differentiate naturally occurring changes (geology, climate, etc.) from changes caused by human impact. If sampling is intended to monitor the effects of a discharge, sample sites upstream and downstream (beyond the mixing zone) of the discharge should be included. Additional sites should be added at distances downstream to assess the extent of the influence of the impact, and any possible recovery. The location of the selected survey unit within a given site should be defined in field notes by reference to map co-ordinates and location relative to permanent bankside objects (e.g. bridges). GPS (global positioning system) receivers may be useful for obtaining accurate co-ordinates. It may also be appropriate to indicate the location of a site by the use of permanent markers such as iron bolts or paint on large stones.
The survey units should all be the same length: 10 m is recommended, but longer lengths may be appropriate under some circumstances. The samples should be collected from the main channel of the river (i.e. the zone that is normally submerged). Rivers with naturally fluctuating discharge have specific algal associations in the flood zone that may be of interest to sample. SIST EN 15708:2010



EN 15708:2009 (E) 12 7.3 Time of sampling The time of sampling depends on the scope of the survey and the local conditions. Seasonal runoff patterns should be considered. It is recommended that surveys are performed and samples collected during periods of stable water flow, preferably at low discharge conditions.
The composition of stream phytobenthos varies throughout the year. A single sample or survey may not be sufficient to characterise fully the diversity in a reach. One sample or survey may, however, be sufficient for spatial and temporal comparisons, as long as all the samples are collected, or surveys performed, at the same time of year. 8 Survey and sampling procedures 8.1 Field survey method: MPS - Macroscopic phytobenthos survey A detailed examination of the entire survey unit should be performed by wading, using an aquascope if observation of the stream bed is hampered by depth or surface turbulence. If some parts are too deep for safe wading, then the survey should cover all areas that can be waded, and a note made. The length of the survey unit shall be recorded. The presence of all phytobenthic growth forms shall be recorded and their abundance estimated. Small specimens should be removed for either bank-side or laboratory examination. The macroscopic growth forms visible with the naked eye may have different appearances, e.g. gelatinous brown cover (often diatoms), green filaments (usually green algae) or dark tufts (red algae or cyanobacteria). The largest filamentous algae may be > 2 m long, whilst some other growth forms may be only 1 mm or 2 mm (e.g. Heribaudiella, Chamaesiphon spp.), and only just visible to the naked eye. Great care shall be taken during this survey as the smallest algal growth forms may be only 1 mm or 2 mm across and easily missed. A checklist of growth forms that may be encountered should be prepared in advance of the survey. If growth forms cannot be classified into one of these classes, then the nature of the growth form should be described (size, colour, appearance) separately on the field record sheet. Additional notes (e.g. recording the size and colour) should also be taken, if appropriate. The abundance estimates should only represent the length of river surveyed (survey unit). The length of the survey unit shall be recorded. NOTE 1 It can be useful to record the identities and abundance of aquatic bryophytes and vascular plants present in the survey unit.
NOTE 2 In some cases (e.g. when monitoring algae capable of causing nuisances), it can only be necessary to record and assess the abundance of one or a few species or growth forms. Samples of those taxa and growth forms that cannot be identified in situ should be collected and examined in a tray (6.1.1.7) filled with some streamwater to get an impression of colour, texture, etc. A hand lens (6.1.1.6) may be useful. Details of sampling procedures are given in 8.2. Those taxa that cannot be identified in the field should be taken back to the laboratory for confirmation. Small quantities of macroscopic algae (and optionally bryophytes) should be retained as voucher specimens.
The abundance of each macroscopic growth form should be estimated using a simple descriptor scale, based on the percentage of the stream or river bed (within the survey unit) that is covered (Table 1). The quantification is based on “qualified judgement”. Scales with about five levels balance the needs for reproducibility and spatial/temporal discrimination. Scales with a greater number of levels may appear to be more accurate but may be less reproducible. SIST EN 15708:2010



EN 15708:2009 (E) 13 Table 1 — Descriptor-scale for abundance estimates of macroscopically visible growth forms Scale Description of coverage at the survey unit 1 Rare: just visible in the field, covers < 1 % of the river bed 2 Occasional: covers 1 % to < 5 % of the river bed 3 Frequent: covers 5 % to < 25 % of the river bed 4 Abundant: covers 25 % to < 50 % of the river bed 5 Dominant: covers > 50 % of the river bed
Depending upon the survey aims, it might be appropriate to use a more precise estimation of abundance. Options include:
 a nine-point scale for es
...